Pregunta básica

Qué célula madre utilizar?

Células madre

Tipos:

EmbrionariasAdultas

hematopoyéticasepitelialesmúsculo cardíacohígadopáncreassistema nervioso

Reprogramadas

Células con capacidad de:

• autoperpetuarse (prolongada o ilimitada)• diferenciarse a distintos tipos celulares

Células madre embrionales

Desventajas

Feeder layer Comportamiento en cultivo muy variable. TumorigénicasRespuesta inmunológica al trasplante

Debate ético: fuente celular

Ventaja

Alta plasticidad: fuente potencial de cualquier célula

Células madre adultas

Desventajas

Menor plasticidad- específicas de linaje

Baja homogeneidad de la muestraDificil acceso (neurales)Baja eficiencia de diferenciación (neurales)

Ventajas

Larga experiencia en células madre hematopoyéticasSin evidencias de tumorigenicidadSin debate ético

Rechazo poco probable (autotrasplante)

Células madre reprogramadas

Reprogramación

Clonación

Clonado de Dolly

Clonado de Dolly

Capacidad de una célula de un linaje de diferenciarse a

células de otro linaje

Transdiferenciación

Transdiferenciación

When the brain turns into bloodVescovi, Science,1999

A: single ROSA26 NSCB: 1 dayC: 8 daysD: neuroesfera (nestin+?)E: neurons (red), astroglia (blue), oligos (green)

Transdiferenciación

A. BM from Balb/c

B. BM from Balb/c transplanted

C. Idem B

D + F. macrophage+ granulocytesE. D + betagal

G + I. macrophages

H. G + beta gal

5 to 12 months after transplantation

to sub-irradiated animals.

H-2kb= ROSA Engraftment: 22 weeks post-transplant

CD3: T cells

CD11: myeloid cells

CD19: B cells

Fusion causes confusion

68 11% newly formed

myocardium

Transdiff= 1/100.000

Cell-cell fusion experiments suggest that the ES cell

dominate the hybrid cell

phenotype. (Clarke and Frisen, Science 2000;

Terada and Scott Nature 2002; Ying and Smith Nature 2002; Cowan and Eggan Science 2005).

The hybrid ES cells possess all

properties of pluripotent stem

cells – including teratoma

formation, self-renewal, yet contains a cell nucleus from

an exogenous cells.

Cowan and Eggan, Science, 2005

Oct3/4Sox2c-MycKlf4

24 candidate factors:Ecat1, Dpp5(Esg1), Fbx015, Nanog, ERas,Dnmt3l, Ecat8, Gdf3, Sox15, Dppa4, Dppa2,

Fthl17, Sall4, Oct4, Sox2, Rex1, Utf1, Tcl1,Dppa3, Klf4, b-cat, cMyc, Stat3, Grb2

2006 - Mouse 2007 - Human

2007 - Human

Nanog

Lin28

Reprogramación células

adultas

VENTAJAS

Pluripotentes

Fácil acceso

Sin dilemas éticos

Rechazo poco probable

Modelos in vitro de enfermedades

DESVENTAJAS

Tumorigenicidad

Baja eficiencia

Carga genética del paciente

Parkinson’s disease (Wernig and Jaenisch, 2008, Maehr and Melton PNAS 2009).

Amyopathic Lateral Sclerosis, (Dimos and Eggan Science 2008)

Type I diabetes (Maehr and Melton PNAS 2009)

ADA-SCID, SBDS, Gaucher disease, Duchenne and Becker Muscular dystrophin, Parkinson’s disease, Huntington disease, JDM, Down syndrome, Lesch-Nyhan syndrome. (Park and Daley Cell 2008).

iPS cells generation from other cell types

• Blood cells (Loh and Daley 2009). B-cells (Hanna and Jaenisch Cell 2008)

• Blood stem cells (Emiinli and Hochedlinger Nat Genet 2009)

• Pancreatic -cells (Stadtfeld and Hochedlinger Cell Stem Cell2008)

• Hepatic and gastric endoderm (Aoi and Yamanaka Science 2008)

• Neural stem cells (Kim and Scholar, Nature 2008)

Human mobilized cells- CD34+

2009 – Human Blood CD34 2011 - Human Blood CD34

2008 – Mouse B Cell

Other tissues: blood.

Peripheral BloodT Cells - Ficoll

2010 – T Cells

2010 – T Cells

2010 – T Cells and CD 34+

Reduced number of transcription factor use: No myc: Nakagawa and Yamanaka, Nat Biotechnol 2008, Wernig and Jaenisch, Cell Stem Cell 2009 No Sox2: by adding GSK-3 inhibitor, Zhou and Ding, Stem cell 2009, in neural stem cell, Kim and Scholer Nature

2008 No Klf4/myc, by addition of Valproic acid : Huangfu and Melton, Nat Biotech 2008 No Myc and Sox2, by addition of BIX01294 and PD0325901 (Zhou and Ding, Cell Stem Cell 2008). Klf4 only by adding Kenpaullone (Lyssiotis and Jaenisch, PNAS 2009)

Specific pathways: TGFb inhibitor replace Sox2 and cMyc and induce Nanog (Maherali and Hochedlinger, Curr Biol 2009, Ichida

and Eggan 2009)

p53 inhibition augments iPS efficiency (Hong and Yamanaka, Nature 2009,Utikal and Hochedlinger Nature 2009, Marion and Blastco Nature 2009, Li and Serrano Nature 2009, Kawamura and Belmonte 2009)

Hypoxia – Yoshida and Yamanaka Cell Stem Cell 2009 WNT signaling stimulates reprogramming efficiency (Marsonm, Jaenisch Cell Stem Cell 2008)

Better vectors: Drug Inducible vectors: Wernig and Jaenisch, Nat Biotechnol 2008, Hockemeyer and Jaenisch, Cell Stem Cell

2008

Non-integrating vectors : adenovirus in hepatocyte (Stadtfeld and Hochedlinger Science 2008) Self-inactivating vectors: Piggy Bac (Yusa and Bradley, Nat Methods 2009) multi-cistronic vectors: single lentiviral cassette ( Carey and Jaenisch, PNAS 2009, Sommer and Mostoslavsky,

Stem Cell 2009) Vector free (episome Yu and Thomson, Science 2009; direct transfection Okita and Yamanaka Science 2008) Direct protein induction: poly arginine modification of recombinant protein (Zhou and Ding, Cell Stem Cell

2009),

2008

2011

Safer hiPSCs – no c-Myc

Easier hiPSCs

Safer hiPSCs – no integration

2009

2009

Safer hiPSCs – no integration

Safer hiPSCs – no integration

hiPSC for genetic diseases??

2007

2011

Terapias regenerativas con células madre

Preguntas básicas

Qué célula madre?

EMBRIONARIA

ADULTA

REPROGRAMADA

hiPSC as a DISEASE MODEL

2008

2009

2008

2009

2008

iPS cells generation in patient fibroblasts

• Parkinson’s disease (Wernig and Jaenisch, 2008, Maehr and Melton PNAS 2009).

• Amyopathic Lateral Sclerosis, (Dimos and Eggan Science 2008)

• Type I diabetes (Maehr and Melton PNAS 2009)

• ADA-SCID, SBDS, Gaucher disease,

Duchenne and Becker Muscular dystrophin,

Parkinson’s disease, Huntington disease,

JDM, Down syndrome, Lesch-Nyhan

syndrome. (Park and Daley Cell 2008).

Gold rush of hiPSCs

FarmacogenomicsIn Vitro

Disease model - challenges

SCIENCE VOL 324 8 MAY 2009

20 anos

???

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

The disease

SPINAL CORD INJURY

Associated with inflammation

Loss of neural connection

Loss of oligodendrocytes

Human embryonic stem cells (hESCs) derived from surplus in vitro fertilized

embryos

Donated for research by the parental donors under informed consent.

The hESC line that is used to produce GRNOPC1 is the H1 line which was

derived before August 9, 2001. GRNOPC1 is a population of living cells

containing oligodendrocyte progenitor cells (OPC).

Studies using this line qualify for U.S. federal research funding, although no

federal funding was received for the development of the product or to

support the clinical trial.

The existing qualified H1 master cell bank of undifferentiated hESCs could

potentially supply sufficient starting material for GRNOPC1 manufacturing to

commercially supply the entire spinal cord injury market in the United States

for more than 20 years.

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

The cells

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

Preclinical studies

Spinal cord- partially injured animals received either no treatment, control cells

or media, or one injection of GRNOPC1 within seven days after injury

Improved:

1. Hind limb locomotor control

2. Paw placement

3. Stride length

4. Remyelination of axons

5. in the injury site

6. Axonal survival and sprouting

Benefits seen up to 9 months after

treatment

Treatment ineffective if administered

more than three months after the injury

due to the scarring that occurs in the

injured cord as part of the inflammatory

response to spinal cord injury.Journal of Neuroscience, Vol. 25, May 2005

Stem Cells and Development, Vol. 15, 2006

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

Preclinical TOXICITY studies

IND (Investigational New Drug) application:

24 separate studies in rats and mice

21,000 pages of data from the animal and in vitro testing of the cells

Absence of Teratoma Formation

Uninjured and spinal cord-injured animals were studied for up to 12 months

after a

single injection of clinical grade GRNOPC1.

No teratomas were found in any animal injected in the spinal cord with clinical

grade GRNOPC1.

Positive control animals in which teratomas were found included animals that

received undifferentiated hESCs or GRNOPC1 preparations that were

intentionally contaminated

with at least 5% of live, undifferentiated hESCs.

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

Preclinical TOXICITY studies

Acute or chronic systemic toxicity

No significant systemic toxicity was found:

Multiple hematology, clinical chemistry, urinalysis, and gross and microscopic

pathology tests were performed on the spinal cord-injured rats that received

GRNOPC1.

GRNOPC1 was not detected outside of the central nervous system in spinal

cord-injected animals.

In some animals, human non-neural differentiated cell types were observed in

the injury site, which did not lead to adverse consequences.

No evidence of allodynia (pain induced by normally non-noxious stimuli) was

detected.

Control and GRNOPC1-treated animals were tested for abnormal behavioral

responses to cold and mechanical stimuli placed at, above or below the area of

injury.

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injuryImmunological studies

In vitro studies were performed to test human allogeneic antibody, T cell and NK

cell responses to GRNOPC1.

GRNOPC1 was incubated with serum and cell samples from normal healthy

volunteers and analyzed for either GRNOPC1 lysis or T cell proliferation.

GRNOPC1 does not have major susceptibility to direct humoral or cell-mediated

alloimmune attack.

These results serve as the rationale for short-term administration of low-dose

immunosuppression in the clinical protocol.

Journal of Neuroimmunology, Vol. 192, 2007

No in vivo data?

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

Preclinical studies- Summary

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

The FDA-approved clinical study is a Phase I multi-center trial designed to

assess the safety and tolerability of GRNOPC1 in patients with complete

ASIA (American Spinal Injury Association) grade A thoracic spinal cord

injuries.

ASIA grading scale - grade A: most severe with complete loss of locomotor

and sensory activity below the site of the injury. Most such patients do not

recover function or respond significantly to physical therapy.

The therapeutic protocol is also limited to subjects with subacute injuries -

injuries that can be treated with GRNOPC1 within seven to 14 days after the

injury (more than 3 months> too late).

The primary endpoint of the study is safety

Clinical Trial

Human embryonic –derived oligodendrocytes for

spinal cord injury

The primary endpoint of the study is safety

Standardized physical examinations and neurological testing will be

administered before and after the injection of GRNOPC1 at specified time

points for one year after the injection

The secondary endpoint of efficacy: return of sensory function or lower

extremity motor function for one year after injection of GRNOPC1.

Subjects will be immune-suppressed from the time of injection with low-dose

tacrolimus for 46 days, at which time the immune suppression will be

tapered and withdrawn at 60 days.

Subjects will be monitored for a total of 15 years after they are

administered GRNOPC1.

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

Clinical- Summary

Scientific

“It would be a disaster, a nightmare, if we ran into …. problems in this very first trial,” said Dr.

John A. Kessler, the chairman of neurology and director of the stem cell institute at

Northwestern University.

Dr. Kessler, whose own daughter was paralyzed from the waist down in a skiing accident,

said he thought Geron’s therapy was not the ideal candidate for the first trial. He said

results showing the therapy worked in moderately injured animals might not apply to more seriously injured people.

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

Public reactions

Political

“I think this approval is directly tied to the change in administration,” said Robert N. Klein,

the chairman of California’s $3 billion stem cell research program. He said he thought the

Bush administration had pressured the F.D.A. to delay the trial.

F.D.A. denies this statement.

Scientific

“We really want the best trial to be done for this first trial, and this might not be it,” Dr.

Kessler said.

The main safety concern is that if raw embryonic cells are put into the body, they can form

tumors. Even though most such tumors do not spread like other cancers, any unwanted growth in the spinal cord can further damage nerves.

“It’s not ready for prime time, at least not in my mind, until we can be assured that the

transplanted stem cells have completely lost the capacity for tumorogenicity,” said Dr.

Steven Goldman, chairman of neurology at the University of Rochester. He was a member

a committee convened by the F.D.A. last April to examine the safety aspects of trials using

therapies from embryonic stem cells.

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

Public reactions

Clinical Trial

Human embryonic –derived oligodendrocytes

for spinal cord injury

Geron, which was formed in 1990 as an antiaging company, is still in the

development stage and is not yet profitable, having lost about $500 million

since its inception. Besides working on stem cells, it is testing drugs for cancer

that influence telomeres, the caps on the ends of chromosomes that help

control the aging of cells. Geron’s market value is about $400 million.

Annual Direct Expenditures

in the U.S. for Selected Diseases

Diseases Expenditures

Diabetes $45,000,000,0001

Alzheimer's $100,000,000,0002

Stroke $30,000,000,0003

Chronic Liver Disease $25,000,000,0004

Spinal Cord Injury $10,000,000,0005

Parkinson's Disease $6,500,000,0006

Terapias regenerativas con células madre

Enfermedades de la sangre

Enfermedades cardíacas

Enfermedades neurodegenerativas

Ciencia

Ciencia-ficción

Turismo de célula madre

Beike Biotech,

China

Las terapias con células madre no son inocuas

No existe evidencia experimental en modelos animalesque sustenten estudios clínicos de terapias regenerativas

para enfermedades neurológicasutilizando células madre hematopoyéticas

La fusión causa confusión y algunos la aprovechan

NUEVO ENSAYO CLÍNICO UTILIZANDO CÉLULAS

MADRE:

GRATUITO

AUTORIZADO POR EL INCUCAI

Terapias regenerativas con células madre en Argentina

Comisión Asesora en células madre y terapias regenerativas

Objetivo: ASESORAMIENTO/INVESTIGACIÓN/DIFUSIÓN

Presidente: Min. Lino Barañao

Coordinadora: Fabiana Arzuaga - H. Cámara de Diputados de la Nación Miembros:Pablo Argibay- Hospital Italiano-CONICETSalvador Bergel- UBARoberto Coco -FECUNDITASAna del Pozo- Hospital GarrahanGustavo Kusminsky- Hospital AustralFlorencia Luna-CONICET-FLACSOJorge Peralta- INCUCAI-UBAFernando Pitossi –Instituto Leloir-CONICETOsvaldo Podhajcer-Instituto Leloir-CONICETPatricia Saidón- Hospital Ramos MejíaMartín Seoane- (ANMAT)Gustavo Sevlever –FLENISusana Sommer – UBA

http://www.mincyt.gov.ar

ISSCR TASK FORCE

www.closerlookatstemcells.org

ISSCR TASK FORCE

www.closerlookatstemcells.org

ISSCR TASK FORCE

www.closerlookatstemcells.org

Visión

Idea de

curación

“instalada”Necesidad de

Terapias

efectivas

Sin efectos

terapéuticos

comprobados

Información

rigurosa

Idea de cura

instalada

Alta

potencialidad

Necesidad de

Terapias

efectivas

Efectos

terapéuticos

comprobados

Información

rigurosa

Visión